Application of Real-Time PCR To Study Effects of Ammonium on Population Size of Ammonia-Oxidizing Bacteria in Soil

作者：Yutaka Okano, Krassimira R. Hristova, Christian M. Leutenegger, Louise E. Jackson, Robert Ford Denison, Binyam Gebreyesus, David Shaner LeBauer, Kate M. Scow · 发表于：Applied and Environmental Microbiology · 年份：2004 · DOI：10.1128/aem.70.2.1008-1016.2004 · 被引用次数：421 · 研究领域：Wastewater Treatment and Nitrogen Removal、Microbial Fuel Cells and Bioremediation、Water Treatment and Disinfection

Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammonia-monooxygenase gene (amoA) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 x 10(5) cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH4)2SO4 application. AOB populations in amended treatments increased from an initial density of approximately 4 x 10(6) cells/g of dry soil to peak values (day 7) of 35 x 10(6) and 66 x 10(6) cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 x 10(9) and 2.2 x 10(9) cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed i...